PROJECT SUMMARY The goal of this project is to define the molecular mechanism by which lung surfactant protein B (SP-B) functions in respiration, and to develop an optimized surfactant mixture that could ultimately be used as a therapeutic for the treatment of Acute Respiratory Distress Syndrome (ARDS). SP-B is the only surfactant protein essential for breathing. It is made in alveolar type II cells as a precursor containing three related domains and is then proteolytically processed into the individual domains (SP-BN, SP-BM, SP-BC), with the middle domain (SP-BM) currently considered to be the ?mature? protein. Surfactant originates from the secretion of lamellar bodies (LB), in which membrane sheets are densely stacked. The exported lipid bilayers then form a lipid monolayer at the air-water interface, which reduces surface tension and facilitates breathing. The exact functions of SP-BM and the other SP-B domains are unknown. Based on crystal structures, biochemical and cell biology experiments, we discovered that SP-BN, which had been largely ignored, is a non-specific lipid transfer protein in lungs. We also found that reconstitution of purified SP-BM into liposomes results in structures that have a striking resemblance to human LBs, suggesting that the entire organelle can be generated from a single protein. Further preliminary results indicate that intratracheal administration of purified SP-BN together with liposomes has a beneficial effect in a mouse model of ARDS. Based on these preliminary results, we will address the following questions: Specific aim #1: What are the functions of SP-BN and SP-BC? We will use purified SP-BN to elucidate the mechanism of lipid transfer and test whether purified SP-BC augments the activity of SP-BN. CRISPR and infection with adeno-associated virus (AAV) constructs will be used to test in mice whether SP-BN has a role in respiration and LB formation. We will use bronchoscopy of ARDS patients and control individuals to test for the presence of SP-BN in lavage fluid. Specific aim #2: What is the function of SP-BM? We will address the mechanism by which SP-BM forms LB-like structures using 2D crystallization and biochemical techniques. We will establish an expression and purification system for SP-BM, which has been a major bottleneck in the field, and determine structures of SP-BM by X-ray crystallography or cryo-EM. We will test whether SP-BN and SP-BC act synergistically with SP-BM to form LB-like structures in vitro. Specific aim #3: Can we generate a surfactant with therapeutic value? We will test whether SP-BN, SP-BM, and possibly SP-BC, together constitute the active extracellular surfactant. Intratracheal administration of a mixture of purified proteins will test whether mice expressing SP- B under an inducible promoter retain normal lung function in the absence of inducer in the diet. The mixture will also be tested in models of ARDS, in which lung injury is induced by either lipopolysaccharide or acid.